PROJECT SUMMARY The improper formation or remodeling of the Pharyngeal Arch Arteries (PAAs) are among some of the most severe and life-threatening forms of congenital heart disease, such as those seen in patients with DiGeorge Syndrome. We have previously shown that the PAA endothelium originates from a subpopulation of mesodermal progenitors termed the Second Heart Field (SHF). In addition, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an RTK which is required for vascular development is highly expressed in these SHF progenitors as they contribute the PAAs. However, to what extent SHF-derived ECs are required for PAA formation, and the mechanisms which regulate the contribution of SHF progenitors to PAAs remains unclear. To address this, we developed a mouse model to conditionally ablate VEGFR2 from the SHF by crossing VEGFR2GFP/+;Isl1Cre/+ and VEGFR2f/f;Rosa26mTmG/mTmG, with the initial hypothesis that the loss of VEGFR2 from SHF vascular progenitors would lead to the absence of PAAs. Unexpectedly, we saw VEGFR2-positive endothelium in VEGFR2GFP/f;Isl1Cre/+ mutant embryos in which VEGFR2 is deleted in the SHF. However, pharyngeal endothelium in these mutants was hypoplastic and disorganized in comparison to control embryos. The use of the Cre reporter Rosa26mTmG demonstrated that these VEGFR2-positive ECs are not of SHF origin but instead, are from another progenitor source, suggesting a possible role for endothelial compensation. Interestingly, embryos heterozygous for VEGFR2 in the SHF lineage showed an impediment in SHF progenitor contribution, in addition to the disorganized and irregular phenotype seen in the knockout embryos, suggesting a possible haploinsufficiency of VEGFR2 in the SHF. Thus, we hypothesize that the SHF contains vascular progenitors essential for proper formation and remodeling of the PAAs. Further, we hypothesize that endothelial cell (EC) progenitors from pre-existing vasculature contributes to the PAAs when SHF contribution is attenuated. Here, we propose to test these hypotheses by addressing the following specific aims: 1) To determine the requirement and mechanisms regulating SHF-derived EC contribution in PAA development; 2) To identify the EC progenitor source(s) that contribute to PAA development when SHF-derived EC contribution is compromised; 3) To determine mechanism(s) that regulate the compensation of PAA ECs when SHF-derived EC contribution is compromised. We will utilize conditional knockout mouse models and immunohistochemistry to fate map and identify the requirement of SHF vascular progenitors in PAA development, and to identify the sources of the non- SHF-derived ECs in the PAAs of embryos null for VEGFR2 in the SHF. Furthermore, we will use a combination of immunohistochemistry and in situ hybridization to provide insight on the mechanisms regulating these vascular progenitors in PAA development. Elucidating sources and mechanisms that drive PAA formation will lead to a better understanding of CHD etiology.